Project Summary A major limitation to understanding the brain is a shortage of technologies for tracking the activity of large populations of individual neurons across multiple layers of synaptic processing. Ideally, these measurements of population activity would be compatible with both optogenetic and chemogenetic manipulations of neural activity to test how targeted perturbations in signal processing alter the input-output relationship of the circuit. The goal of this proposal is to develop a technology that combines (1) large-scale population calcium imaging via light sheet microscopy, (2) large-scale microelectrode array (MEA) measurements, and (3) visual stimulation of neurons. We will use this system to measure the complete set of feedforward transformation in a major model neural circuit, the mammalian retina. Specifically, we will control the activity of photoreceptors with light, calcium image the resultant activity of bipolar cells (processing layer) and use an MEA to measure spiking activity of retinal ganglion cells (output layer). Developing this system is the single technical hurdle for a future BRAIN Initiative directed R01 application that will employ this system along with cell-type specific chemogenetic inactivation of inhibitory interneurons to determine how diverse cell types shape circuit function. The rationales for developing this system for retina are: (1) the retina is photosensitive, which allows the input to the circuit to be controlled by light; (2) the retina balances simplicity and complexity with two layers of synaptic processing and dozens of parallel information processing pathways built out of ~80 cell types; and (3) nearly all blinding conditions are diseases of the retina, thus understanding the neurophysiology of the retina is the best bet to treating blindness. The primary significance of this proposal is that it will produce a system that allows the signal transformations across multiple synaptic layers to be measured simultaneously. This is significant because the transformations that occur across synapses are nonlinear and are strongly shaped by noise correlations within the circuit, necessitating simultaneous measurements. The primary innovation of this proposal is that it will produce the first system to synthesize technologies for simultaneous cellular resolution targeting of neurons for visual stimulation, calcium imaging, and large-scale MEA measurements. This synthesis will advance our capability to generate a complete understanding of a neural circuit. In a future application, we will use this technology to determine how the neural signals are transformed across the retina to form functionally distinct encoding pathways, and how this transformation is shaped by inhibitory interneurons.